Compositions and methods for treating kidney disease

ABSTRACT

The present invention relates to methods and compositions for the prevention and treatment of renal damage. The invention provides protein-based renal therapeutic agents for administration to subjects in order to prevent or treat renal degeneration or damage.

The present application is a CIP of U.S. application Ser. No. 11/508,701filed Aug. 23, 2006, which claims priority to U.S. ProvisionalApplication Ser. No. 60/710,803 filed Aug. 23, 2005, which claims thebenefit of U.S. patent application Ser. No. 10/464,368 filed Jun. 16,2003, which claims the benefit of International Patent ApplicationSerial No. PCT/US2003/019260 filed Jun. 16, 2003, which claims priorityto U.S. Provisional Application No. 60/388,970 filed Jun. 14, 2002.

BACKGROUND OF INVENTION

A. Field of the Invention

The present invention relates to compositions and methods for theprevention and treatment of renal damage. In particular, the inventionrelates to administration of novel therapeutics to subjects in order toprevent or treat renal degeneration or damage. These novel therapeuticsinclude antibodies, peptides, and small molecules based upon theWISE/SOST family of proteins.

B. Background of the Invention

The mammalian renal system serves primary roles both in the removal ofcatabolic waste products from the bloodstream and in the maintenance offluid and electrolyte balances in the body. Renal failures are,therefore, life-threatening conditions in which the build-up ofcatabolites and other toxins, and/or the development of significantimbalances in electrolytes or fluids, may lead to the failure of othermajor organs systems and death. Chronic renal failure is a debilitatingand life-threatening disease for which no adequate treatment exists.

Tubular damage and interstitial fibrosis are the final common pathwaysleading to end stage renal disease. Irrespective of the nature of theinitial renal injury, the degree of tubular damage parallels theimpairment of renal function. Once nephronic degeneration or tubulardamage is established, it cannot be reversed or repaired by currentlyavailable treatment, and renal function deteriorates to renal failure,which is often life threatening. Renal damage and failure can only bemanaged through dialysis or organ transplantation.

Dialysis dependency is one of the leading causes of morbidity andmortality in the world. Despite advancement in understanding thepathophysiology of renal diseases, the incidence of end-stage renaldisease is increasing. Approximately 600 patients per million receivechronic dialysis each year in the United States, at an average costapproaching $60,000-$80,000 per patient per year. Of the new cases ofend-stage renal disease each year, approximately 28-33% are due todiabetic nephropathy (or diabetic glomerulopathy or diabetic renalhypertrophy), 24-29% are due to hypertensive nephrosclerosis (orhypertensive glomerulosclerosis), and 15-22% are due toglomerulonephritis. The 5-year survival rate for all chronic dialysispatients is approximately 40%, but for patients over 65, the rate dropsto approximately 20%.

A need remains, therefore, for treatments that will prevent theprogressive loss of renal function which has caused almost two hundredthousand patients in the United States alone to become dependent uponchronic dialysis, and which results in the premature deaths of tens ofthousands each year.

SUMMARY OF INVENTION

The present invention provides protein-based renal therapeutic agentsfor administration to subjects in, or at risk of, renal failure. Themethods and compositions of the present invention may be used toprevent, inhibit, delay, or reverse nephronic degeneration, whichotherwise leads to the need for renal replacement therapy to preventdeath. Specifically, the present invention is directed to compositionsand methods that regulate the interaction between SOST and WISE proteinswith their natural receptors. Exemplary natural receptors for WISE andSOST proteins include, but are not limited to, LRP5, LRP6, and BMPmolecules. Methods and compositions of the present invention thereforeprovide a therapy that may reverse nephronic degeneration and/or preventthe progressive loss of renal function, thereby preventing prematuredeath.

Methods of the invention include administering a therapeuticallyeffective amount of an antibody to a patient in which the antibodyspecifically binds a peptide having at least 75%, 80%, 85%, 90%, 95%,99% or more identity to at least 5, 8, 10, 15, 20 or more contiguousamino acids of a developmental regulator and the antibody interfereswith the interaction between at least two developmental regulatorsthereby providing nephron protection and/or regeneration. Exemplarydevelopmental regulators include those molecules disclosed as SEQ IDNOS: 1-217.

In some embodiments of the invention, the developmental regulators are aligand and the ligand's natural receptor. For example, the ligand may beWISE and a known WISE receptor, such as LRP5, LRP6, BMP2, or BMP7.Another exemplary pair is SOST protein and one or more of its knownreceptors, e.g., LRP5, LRP6, BMP6, or BMP7.

The invention also provides a pharmaceutical composition foradministration to a subject that includes an antibody and optionalexcipient(s). Antibodies suitable for the present invention may beadministered in a therapeutically effective amount resulting in animprovement of renal function by at least 10%, 15%, 20%, 25%, or morefollowing renal insult, as measured by a standard assay of renalfunction. Examples of such assays are provided herein. For example, asuitable assay of renal function include, determining rates of increasein Blood Urea Nitrogen (BUN) levels, rates of increase in serumcreatinine, static measurements of BUN, static measurements of serumcreatinine, glomerular filtration rates (GFR), ratios of BUN/creatinine,and serum concentrations of sodium (Na+). Suitable excipients include,but are not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof.

Antibodies of the invention may be monoclonal, polyclonal, humanized, ora fragment thereof (Fab or Fab₂), as described in greater detail, below.Preferably, antibodies of the present invention specifically bind apeptide having at least 75%, 80%, 85%, 90%, 95%, 99% or more identity toat least 5, 8, 10, 15, 20 or more contiguous amino acids of adevelopmental regulator and the antibody interferes with the interactionbetween at least two developmental regulators thereby providing nephronprotection and/or regeneration. More preferably, the antibodyspecifically binds a peptide having at least 75%, 80%, 85%, 90%, 95%,99% or more identity to at least 5, 8, 10, 15, 20 or more contiguousamino acids of or encoded by SEQ ID NOS. 2, 4, 6, 8, 10, 12, 14, 15-18,20, 85-87, 91, 93, 95, 98, 101, 103, 105, and 109-217; preferably SEQ IDNOS. 90-108, 215, and 216; more preferably, SEQ ID NOS. 19-89, 15-18,and 217; preferentially, SEQ ID NOS. 90-93, 215, and 216; alternatively,SEQ ID NOS. 15-20 and 217; more preferably, SEQ ID NOS. 92, 93, and 215;more preferably SEQ ID NOS. 15-18 and 217; ideally, SEQ ID NOS. 15-18.Alternatively, the antibody specifically binds a peptide having at least75%, 80%, 85%, 90%, 95%, 99% or more identity to at least 5, 8, 10, 15,20 or more contiguous amino acids of positions 50-62, 68-80, or 83-98 ofSEQ ID NOS. 20, and 215-217.

The method and pharmaceutical composition of the invention may beadministered to any subject receiving renal injury, chemical or physicalinsult resulting in apoptosis or necrosis of renal tissue, disease, orthose otherwise at risk of chronic renal failure. For example, subjectsin, or at risk of, chronic renal failure, or at risk of the need forrenal replacement therapy, include but are not limited to the following:subjects which may be regarded as afflicted with chronic renal failure,end-stage renal disease, chronic diabetic nephropathy, hypertensivenephrosclerosis, chronic glomerulonephritis, hereditary nephritis,and/or renal dysplasia; subjects having a biopsy indicating glomerularhypertrophy, tubular hypertrophy, chronic glomerulosclerosis, renal cellcarcinoma, and/or chronic tubulointerstitial sclerosis; subjects havingan ultrasound, MRI, CAT scan, or other non-invasive examinationindicating renal fibrosis.

The methods and compositions of the present invention may be utilizedfor any mammalian subject. For example, human subjects or patients,domesticated mammals (e.g., dogs, cats, horses), mammals withsignificant commercial value (e.g., dairy cows, beef cattle, sportinganimals), mammals with significant scientific value (e.g., captive orfree specimens of endangered species), or mammals which otherwise havevalue.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd Ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

As used herein, “antibody” includes reference to an immunoglobulinmolecule immunologically reactive with a particular antigen, andincludes both polyclonal and monoclonal antibodies. The term alsoincludes genetically engineered forms such as chimeric antibodies (e.g.,humanized murine antibodies) and heteroconjugate antibodies (e.g.,bispecific antibodies). The term “antibody” also includes antigenbinding forms of antibodies, including fragments with antigen-bindingcapability (e.g., Fab′, F(ab′)₂, Fab, Fv and rIgG). See also, PierceCatalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.).See also, e.g., Kuby, J., Immunology, 3^(rd) Ed., W.H. Freeman & Co.,New York (1998). The term also refers to recombinant single chain Fvfragments (scFv). The term antibody also includes bivalent or bispecificmolecules, diabodies, triabodies, and tetrabodies. Bivalent andbispecific molecules are described in, e.g., Kostelny et al. (1992) JImmunol 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579,Hollinger et al., 1993, supra, Gruber et al. (1994) J Immunol: 5368, Zhuet al. (1997) Protein Sci 6:781, Hu et al. (1996) Cancer Res. 56:3055,Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995)Protein Eng. 8:301.

An antibody immunologically reactive with a particular antigen can begenerated by recombinant methods such as selection of libraries ofrecombinant antibodies in phage or similar vectors, see, e.g., Huse etal., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546(1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or byimmunizing an animal with the antigen or with DNA encoding the antigen.

The term “insult” refers to any injury or damage to a cell or populationof cells that results in cell death or apoptosis, necrosis, alteredkidney function, or decreased kidney function. An insult may have avariety of causes including, but not limited to, disease, chemicalinjury, or physical injury.

The phrase “specifically binds” when referring to a protein or peptide,refers to a binding reaction that is determinative of the presence ofthe protein, in a heterogeneous population of proteins and otherbiologics. Thus, under designated immunoassay conditions, the specifiedantibodies bind to a particular protein sequence at least two times thebackground and more typically more than 10 to 100 times background.

Specific recognition by an antibody under such conditions requires anantibody that is selected for its specificity for a particular protein.For example, antibodies raised against a particular protein, polymorphicvariants, alleles, orthologs, and conservatively modified variants, orsplice variants, or portions thereof, can be selected to obtain onlythose polyclonal antibodies that are specifically immunoreactive withWISE/SOST-like peptides such as those exemplified by SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 15-18, 20-82, 85-87, 91, 93, 95, 98, 101, 103, 105,109-217 and not with other random proteins. This selection may beachieved by subtracting out antibodies that cross-react with othermolecules. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988) for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same asmeasured using a BLAST or BLAST 2.0 sequence comparison algorithms withdefault parameters described below, or by manual alignment and visualinspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/or the like). Such sequences are said to be “substantially identical”when they have about 60% identity, preferably 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, once compared and aligned for maximum correspondenceover a comparison window or designated region. This definition alsorefers to, or may be applied to, the complement of a test sequence. Thedefinition also includes sequences that have deletions and/or additions,as well as those that have substitutions, as well as naturallyoccurring, e.g., polymorphic or allelic variants, and man-made variants.As described below, the preferred algorithms can account for gaps andthe like.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof one of the number of contiguous positions selected from the grouptypically of from 20 to 600, usually about 50 to about 200, more usuallyabout 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

Preferred examples of algorithms that are suitable for determiningpercent sequence identity and sequence similarity include the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990). BLAST and BLAST 2.0 are used, with the parameters describedherein, to determine percent sequence identity for the nucleic acids andproteins of the invention. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/). This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, e.g.,for nucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001. Log valuesmay be large negative numbers, e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110,150, 170, etc.

The phrase “conditions suitable for protein binding” refers to thoseconditions (in terms of salt concentration, pH, detergent, proteinconcentration, temperature, etc.) which allow for binding to occurbetween a protein and its binding partner in solution. The conditionsare not so lenient that a significant amount of nonspecific proteinbinding occurs.

As used herein, the term “developmental regulators” refers to moleculesassociated with the Wnt and BMP signaling pathways. Specifically, theterm refers to the ligands and receptors responsible for regulating theWnt and BMP signaling pathways including, but not limited to, LRP5,LRP6, BMP2, BMP4, BMP6, and BMP7. For example, several of thesedevelopmental regulators are provided by SEQ ID NOs: 1-217 as presentedin the present application.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 graphically illustrates inhibition of SOST association with LRP6.Relative LRP6 binding to variants of SOST was measured followingimmunoprecipitation. SOST variants M1, M2, M3, and M8 significantlyexhibited reduced binding to LRP6 compared to wild type SOST.

DETAILED DESCRIPTION I. Introduction

The present invention provides compositions and methods of using certainprotein-based renal therapeutic agents that surprisingly prevent,inhibit, delay or alleviate the progressive loss of renal function. In apreferred embodiment, the present invention is suitable for treatment ofrenal disease.

In some forms, renal disease is caused by aberrant signal transductionduring kidney development. The kidney develops from the ureteric bud,extending out from a pre-existing epithelial tube, giving rise to thebranched collecting duct system while the surrounding metanephricmesenchyme undergoes mesenchymal-epithelial transition to form theproximal parts of the nephron. Signaling by members of the Wnt, BMP andFGF protein families, mediate this nephrogenesis by adjusting thebalance between the ureteric bud epithelium, stromal and nephrogenictissues. Inappropriate alteration of the balance of these signalingpathways, gives rise to renal disease. For example, over-activation ofthe Wnt pathway leads to cancer development (e.g. Wilms tumor), whileinhibition of BMP signaling results in nephronic degeneration, bothultimately leading to renal failure.

WISE and/or SOST signaling also influences mature kidney tissuehomeostasis, particularly in the case of renal damage or disease. Incertain embodiments of the present invention, renal disease or damage ismitigated or reversed by administering to a patient antibodies thatperturb or block the association of WISE and/or SOST to its receptormolecules in vivo. For example, administration of antibodies that mimicthe WISE and/or SOST association with LRP5 or LRP6 may be used to subdueover-activated Wnt signaling in the treatment of kidney cancer.Alternatively, the association of WISE and/or SOST with BMP6, BMP7,and/or BMP2 may be inhibited to allow BMP signaling, which may result inprotection from nephronic injury and/or promotion of nephronicregeneration.

II. Biological Assays of the Invention

The phrase “nephronic degeneration” refers to deterioration of anindividual's kidney in which kidney or renal function is diminished asresult of tissue necrosis or apoptosis by at least 5% preferably 10%,15%, 20%, 25%, 30%, 40% 50% or more from the range of normal valuesmedically determined for the individual. Nephronic degeneration canresult from physical insult, chemical insult, or disease. The presenceof nephronic degeneration can be measured by assays well known to thoseof ordinary skill of the art, such as elevation of serum creatininelevels or decrease in creatinine clearance (see, Brenner and Lazarus(1994), in HARRISON'S PRINCIPLES OF INTERNAL MEDICINE, 13th edition,Isselbacher et al., eds., McGraw Hill Text, N.Y.). Preferably a decreaseof 5%, more preferably 10%, 15%, 20%, 25%, 30%, 40%, 50% or more ofcreatinine clearance compared to normal levels marks nephronicdegeneration. Likewise, a 5% elevation of serum creatinine levels, morepreferably 10%, 15%, 20%, 25%, 30%, 40%, 50% or more compared to normallevels indicates nephronic degeneration.

The phrase “nephron protection” refers to an in vivo phenomenon thatprotects against and prevents degeneration of nephronic or renalfunction caused by physical insult, chemical insult, or disease. Assuch, nephron protection refers to an in vivo phenomenon that inhibitselevation of serum creatinine levels or decrease in creatinine clearanceby at least 5% preferably 10%, 15%, 20%, 25%, 30%, 40% 50% or more fromthe range of elevated values medically determined for the individual.Nephron protection also encompasses regeneration or repair of degeneratenephronic function caused by tissue necrosis or apoptosis resulting fromphysical insult, chemical insult, or disease. The regeneration or repairof degenerate nephronic function can be measured by assays well known tothose of ordinary skill of the art, such as serum creatinine levels orcreatinine clearance. Preferably an increase of 5%, more preferably 10%,15%, 20%, 25%, 30%, 40%, 50% or more of creatinine clearance compared tonormal levels marks nephronic protection. Likewise, a 5% decrease ofserum creatinine levels, more preferably a 10%, 15%, 20%, 25%, 30%, 40%,50% or more compared to normal levels indicates nephronic protection.

Assays of renal function are well known to those of ordinary skill ofthe art and include, without being limited to, rates of increase inBlood Urea Nitrogen (BUN) levels, rates of increase in serum creatinine,static measurements of BUN, static measurements of serum creatinine,glomerular filtration rates (GFR), ratios of BUN/creatinine, serumconcentrations of sodium (Na+), urine/plasma ratios for creatinine,urine/plasma ratios for urea, urine osmolality, daily urine output, andthe like (see, Brenner and Lazarus (1994), in HARRISON'S PRINCIPLES OFINTERNAL MEDICINE, 13th edition, Isselbacher et al., eds., McGraw HillText, N.Y.). Exemplary normal levels are as follows: serum creatininelevels of 0.8 to 1.4 mg/dL; BUN levels of 5 to 20 mg/dL; GFR score of 90mL/min or more; BUN/Creatinine ratio of 10:1 to 20:1 and up to 30:1 ininfants under 12 months of age; and serum sodium levels of 135 to 145mEq/L. A skilled artisan will recognize that the normal ranges may varywith age, muscle mass, gender, weight, body surface area, and othercharacteristics. An “improvement” in one of the assays of renal functionrefers to an increase or decrease in level that is closer to the normalrange. For example, a 10% improvement of a serum creatinine level of 0.2mg/dL would be a serum creatinine level of 0.22 mg/dL, while a 10%improvement of a serum creatinine level of 3.0 mg/dL would be a serumcreatinine level of 2.7 mg/dL.

III. Therapeutic Compositions

The present invention is directed to compositions and methods thatregulate the interaction between SOST and WISE proteins with theirnatural receptors, particularly LRP5, LRP6, and BMP molecules. The renaltherapeutic agents of the invention include, but are not limited to,peptides, proteins, antibodies, and small molecules derived from theWISE/SOST and LRP/BMP families and resultantly regulate Writ and BMPsignaling. For example, any peptide of at least 20, preferably 25, 30,35, 40, 50 or more amino acids encoded by SEQ ID NO: 2, 4, 6, 8, 10, 12,14, 15-18, 20-82, 85-87, 91, 93, 95, 98, 101, 103, 105, 109-217, or anyfragment of any sequence thereof, may be used to raise antibodies,derive peptides, or derive small molecules suitable for antagonizing theinteraction between SOST and WISE proteins with their natural receptors.

Such peptides may provide the basis of therapeutics by their inherentproperties. For example, as inhibitors of renal damage, blockingpeptides that antagonize the interaction between SOST and WISE proteinswith their natural receptors may be useful. Further, peptides thatactivate SOST and WISE natural receptors by mimicking the necessaryinteraction between SOST or WISE and their natural receptors may also beuseful. Exemplary antagonizing or activating peptides may include thoseprovided by SEQ ID NOS: 21-82 or fragments of SEQ ID NOS: 2, 4, 6, 8,10, 12, 14, 15-18, 20, 85-87, 91, 93, 95, 98, 101, 103, 105, 109-217.

A. Peptides and Proteins

Proteins and peptides useful to the invention may be isolated fromnatural sources, prepared synthetically or recombinantly, or anycombination of the same using techniques well known to those of skill inthe art. Generally, any purification protocol suitable for isolatingproteins and known to those of skill in the art can be used. Forexample, affinity purification, column chromatography techniques,precipitation protocols and other methods for separating proteins may beused (see, e.g., Scopes, Protein Purification: Principles and Practice(1982); and U.S. Pat. No. 4,673,641). Further, peptides may be producedsynthetically using solid phase techniques and other techniques known tothose skilled in the art (see, Barany, G. and Merrifield, R. B. SolidPhase Peptide Synthesis in PEPTIDES, Vol. 2, Academic Press, New York,N.Y., pp. 100-118 (1980)). Peptides and proteins of the invention mayalso be available commercially, or may be produced commercially.

B. Antibodies

The renal therapeutic agents of the present invention may be antibodiesthat recognize developmental regulator proteins, polypeptides, aminoacid sequences, or fragments thereof. Suitable antibodies include thosethat recognize the WISE/SOST and LRP/BMP families and resultantlyregulate Wnt and BMP signaling, such as those described in U.S.application Ser. No. 11/508,701 and incorporated herein by reference.For example, antibodies of the invention will recognize proteins oramino acid sequences encoding developmental regulators or fragmentsthereof, such as, but not limited to, those provided by SEQ ID NOS: 2,4, 6, 8, 10, 12, 14, 15-18, 20-82, 85-87, 91, 93, 95, 98, 101, 103, 105,109-217. More preferably, the antibody specifically binds a peptidehaving at least 75%, 80%, 85%, 90%, 95%, 99% or more identity to atleast 5, 8, 10, 15, 20 or more contiguous amino acids of or encoded bySEQ ID NOS. 2, 4, 6, 8, 10, 12, 14, 15-18, 20, 85-87, 91, 93, 95, 98,101, 103, 105, and 109-217; more preferably SEQ ID NOS. 90-108, 215, and216; more preferably, SEQ ID NOS. 19-89, 15-18, and 217; morepreferably, SEQ ID NOS. 90-93, 215, and 216; more preferably, SEQ IDNOS. 15-20 and 217; more preferably, SEQ ID NOS. 92, 93, and 215; morepreferably SEQ ID NOS. 15-18 and 217; more preferably, SEQ ID NOS.15-18. Alternatively, the antibody specifically binds a peptide havingat least 75%, 80%, 85%, 90%, 95%, 99% or more identity to at least 5, 8,10, 15, 20 or more contiguous amino acids of positions 50-62, 68-80, or83-98 of SEQ ID NOS. 20, and 215-217.

When the above family of amino acid sequences, including WISE and SOST,are allowed to bind to their natural receptors, renal regeneration isrepressed. When the above-mentioned family of amino acid sequences areprevented from binding to their natural receptors, renal regenerationwill increase. Thus, the present invention relates to tools and methodsused to inhibit or mimic the binding of the WISE/SOST family to theirnatural receptors.

1. Antigen Specificity and Production

The present invention provides at least one antibody that inhibitsinteraction between Wnt or BMP antagonistic ligands (developmentalregulators) with LRP or BMP receptors, thus promoting constitutive Wntor BMP signaling and renal regeneration. Suitable antibodies areobtained by immunizing a host animal with peptides, or antigens, thatare all or a portion of the subject protein of the presently claimedinvention. The antigen may be the complete protein, or fragments andderivatives thereof. For example, a suitable antigen may have at least75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to at least 5, 8, 10,12, 15, 20, or 25 contiguous amino acids of a protein encoded by SEQ IDNOS: 2, 4, 6, 8, 10, 12, 14, 15-18, 20-82, 85-87, 91, 93, 95, 98, 101,103, 105, 109-217. More preferably, the antibody specifically binds apeptide having at least 75%, 80%, 85%, 90%, 95%, 99% or more identity toat least 5, 8, 10, 15, 20 or more contiguous amino acids of or encodedby SEQ ID NOS. 2, 4, 6, 8, 10, 12, 14, 15-18, 20, 85-87, 91, 93, 95, 98,101, 103, 105, and 109-217; more preferably SEQ ID NOS. 90-108, 215, and216; more preferably, SEQ ID NOS. 19-89, 15-18, and 217; morepreferably, SEQ ID NOS. 90-93, 215, and 216; more preferably, SEQ IDNOS. 15-20 and 217; more preferably, SEQ ID NOS. 92, 93, and 215; morepreferably SEQ ID NOS. 15-18 and 217; more preferably, SEQ ID NOS.15-18. Alternatively, the antibody specifically binds a peptide havingat least 75%, 80%, 85%, 90%, 95%, 99% or more identity to at least 5, 8,10, 15, 20 or more contiguous amino acids of positions 50-62, 68-80, or83-98 of SEQ ID NOS. 20, and 215-217.

Some exemplary embodiment of the present invention includes antibodiesthat inhibit, block, or otherwise interfere with the specific binding ofan LRP or BMP molecule to a Wnt or BMP antagonistic ligand. A skilledartisan will recognize that an antigen may be selected to generate anantibody that interferes by specifically binding to the LRP or BMPmolecule or by specifically binding to the Wnt or BMP antagonisticligand. The selected antigen will result in an antibody that willspecifically bind to WISE-like or SOST-like proteins and prevent theinteraction of WISE-like or SOST-like proteins with LRP5, LRP6, BMP2,BMP6, or BMP7. in alternative examples, a selected antigen will resultin an antibody that will specifically bind to LRP5, LRP6, or BMPmolecules and prevent the interaction with WISE-like or SOST-likeproteins.

Suitable amounts of well-characterized antigen for production ofantibodies can be obtained using standard techniques known in the artsuch as, but not limited to, cloning or synthetic synthesis. Antigenicproteins can be obtained from transfected cultured cells thatoverproduce the antigen of interest. For example, expression vectorsthat have nucleotide sequences encoding an antigen of interest can beconstructed, transfected into cultured cells, and then the antigen canbe subsequently isolated using methods well-known to those skilled inthe art (see, Wilson et al., J. Exp. Med. 173:137, 1991; Wilson et al.,J. Immunol. 150:5013, 1993). Alternatively, DNA molecules encoding anantigen of choice can be obtained by synthesizing DNA molecules usingmutually priming long oligonucleotides (see, Ausubel et al., (eds.),Current Protocols In Molecular Biology, pages 8.2.8 to 8.2.13, 1990;Wosnick et al., Gene 60:115, 1987; and Ausubel et al. (eds.), ShortProtocols In Molecular Biology, 3rd Edition, pages 8-8 to 8-9, JohnWiley & Sons, Inc., 1995). As a skilled artisan will recognize,established techniques using the polymerase chain reaction provide theability to synthesize antigens (Adang et al., Plant Molec. Biol.21:1131, 1993; Bambot et al., PCR Methods and Applications 2:266, 1993;Dillon et al., “Use of the Polymerase Chain Reaction for the RapidConstruction of Synthetic Genes,” in METHODS IN MOLECULAR BIOLOGY, Vol.15: PCR PROTOCOLS: CURRENT METHODS AND APPLICATIONS, White (ed.), pages263 268, Humana Press, Inc. 1993). Once produced, the antigen of choiceis used to generate antigen specific antibodies.

2. Antibody Production

The present invention provides antibodies as renal therapeutic agents.It is envisioned that such antibodies include, but are not limited to,polyclonal, monoclonal, humanized, part human, or fragments thereof. Askilled artisan will appreciate the benefits and disadvantages of thetype of antibody used for therapeutic treatment and will furtherrecognize the selection is dependent upon the intended use.

a. Polyclonal Antibodies

Means for preparing and characterizing polyclonal antibodies are wellknown to those skilled in the art (see, e.g., Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1988; incorporated herein byreference). For example, for the preparation of polyclonal antibodies,the first step is immunization of the host animal with the targetantigen, where the target antigen will preferably be in substantiallypure form, with less than about 1% contaminant. The antigen may includethe complete target protein, fragments, or derivatives thereof. Toprepare polyclonal antisera an animal is immunized with an antigen ofinterest, and antisera is collected from that immunized animal. A widerange of animal species can be used for the production of antisera.Typically the animal used for production of anti-antisera is a rabbit,mouse, rat, hamster, guinea pig or goat. Because of the relatively largeblood volume of rabbits, a rabbit is a preferred choice for theproduction of polyclonal antibodies.

The amount of antigen used in the production of polyclonal antibodiesvaries upon the nature of the antigen as well as the animal used forimmunization. A variety of routes can be used to administer the antigenof choice; subcutaneous, intramuscular, intradermal, intravenous,intraperitoneal and intrasplenic. The production of polyclonalantibodies may be monitored by sampling blood of the immunized animal atvarious points following immunization. A second, booster injection, mayalso be given. The process of boosting and titering is repeated until asuitable titer is achieved. When a desired titer level is obtained, theimmunized animal can be bled and the serum isolated and stored. Theanimal can also be used to generate monoclonal antibodies, as is wellknown to those skilled in the art.

The immunogenicity of a particular composition can be enhanced by theuse of non-specific stimulators of the immune response, known asadjuvants. Exemplary adjuvants include complete Freund's adjuvant, anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis; incomplete Freund's adjuvant; and aluminumhydroxide adjuvant.

It may also be desired to boost the host immune system, as may beachieved by associating the antigen with, or coupling the antigen to, acarrier. Exemplary carriers include keyhole limpet hemocyanin (KLH) andbovine serum albumin (BSA). Other albumins such as ovalbumin, mouseserum albumin or rabbit serum albumin can also be used as carriers. Asis also known in the art, a given composition may vary in itsimmunogenicity.

b. Monoclonal Antibodies

Monoclonal antibodies (blabs) may be readily prepared through use ofwell-known techniques to those skilled in the art, such as thoseexemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with the selected antigen. The antigen is administered in amanner effective to stimulate antibody-producing cells. Rodents such asmice and rats are preferred animals, however, the use of rabbit, sheepand frog cells is also possible.

By way of example, following immunization the somatic cells with thepotential for producing antigen specific antibodies, specifically Blymphocytes (B cells), are selected for use in the MAb generatingprotocol. These cells may be obtained from biopsied spleens, tonsils orlymph nodes, or from a peripheral blood sample. Spleen cells andperipheral blood cells are preferred, the former because they are a richsource of antibody-producing cells that are in the dividing plasmablaststage, and the latter because peripheral blood is easily accessible.Often, a panel of animals will have been immunized and the spleen of theanimal with the highest antibody titer will be removed and the spleenlymphocytes obtained by homogenizing the spleen with a syringe.Typically, a spleen from an immunized mouse contains approximately 5×10⁷to 2×10⁸ lymphocytes.

The anti-antigen antibody-producing B lymphocytes from the immunizedanimal are then fused with cells of an immortal myeloma cell, generallyone of the same species as the animal that was immunized. Myeloma celllines suited for use in hybridoma-producing fusion procedures preferablyare non-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65 66, 1986; Campbell, pp. 75 83, 1984;each incorporated herein by reference). For example, where the immunizedanimal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 41,Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; forrats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F, 4B210 or one of theabove listed mouse cell lines; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6, are all useful in connection with human cell fusions.

The heterogeneous cell population may be cultured in the presence of aselection medium to select out the hybridoma cells. A suitable selectionmedium includes an inhibitor of de novo synthesis, such as aminopterinin HAT medium, methotrexate in HMT medium, or azaserine in AzaH mediumplus the necessary purine and/or pyrimidine salvage precursors (i.e.hypoxanthine and thymidine in HAT or HMT media; hypoxanthine in AzaHmedium). Only cells capable of operating nucleotide salvage pathways areable to survive in the selection medium. The myeloma cells are defectivein key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyltransferase (HPRT), and cannot survive. The B cells can operate thispathway, but they have a limited life span in culture and generally diewithin about two weeks. Therefore, the only cells that can survive inthe selective media are those hybrids formed from myeloma and B cells(hybridomas).

Culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired anti-antigen reactivity. The assayshould be sensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual anti-antigen antibody-producing cell lines, which clones canthen be propagated indefinitely to provide MAbs. The cell lines may beexploited for MAb production in two basic ways. A sample of thehybridoma can be injected (often into the peritoneal cavity) into ahistocompatible animal of the type that was used to provide the somaticand myeloma cells for the original fusion. The injected animal developstumors secreting the specific monoclonal antibody produced by the fusedcell hybrid. The body fluids of the animal, such as serum or ascitesfluid, can then be tapped to provide MAbs in high concentration. Theindividual cell lines could also be cultured in vitro, where the MAbsare naturally secreted into the culture medium from which they can bereadily obtained in high concentrations.

MAbs produced by either means will generally be further purified, e.g.,using filtration, centrifugation and various chromatographic methods,such as HPLC or affinity chromatography, all of which purificationtechniques are well known to those of skill in the art. Thesepurification techniques each involve fractionation to separate thedesired antibody from other components of a mixture. Analytical methodsparticularly suited to the preparation of antibodies include, forexample, protein A-Sepharose and/or protein G-Sepharose chromatography.

c. Humanized Antibodies

Also of interest are humanized antibodies. Methods of humanizingantibodies are known in the art. The humanized antibody may be theproduct of an animal having transgenic human immunoglobulin constantregion genes (see for example International Patent Applications WO90/10077 and WO 90/04036, both incorporated herein by reference).Alternatively, the antibody of interest may be engineered by recombinantDNA techniques to substitute the CH1, CH2, CH3, hinge domains, and/orthe framework domain with the corresponding human sequence (see WO92/02190 and incorporated herein by reference).

The use of 1 g cDNA for construction of chimeric immunoglobulin genes isknown in the art (Liu et al. P.N.A.S. 84:3439, 1987 and incorporatedherein by reference). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA. The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(see U.S. Pat. Nos. 4,683,195 and 4,683,202, both incorporated herein byreference). Alternatively, a library is made and screened to isolate thesequence of interest. The DNA sequence encoding the variable region ofthe antibody is then fused to human constant region sequences. Thesequences of human constant region genes may be found in Kabat et al.Sequences of Proteins of Immunological Interest, N.I.H. publication no.91-3242, 1991 and incorporated herein by reference. Human C region genesare readily available from known clones. The chimeric, humanizedantibody is then expressed by conventional methods known to those ofskill in the art.

d. Antibody Fragments

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g. by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.The following patents and patent applications are specificallyincorporated herein by reference for the preparation and use offunctional, antigen-binding regions of antibodies, including scFv, Fv,Fab′, Fab and F(ab′)₂ fragments: U.S. Pat. Nos. 5,855,866; 5,965,132;6,051,230; 6,004,555; and 5,877,289.

Also contemplated are diabodies, which are small antibody fragments withtwo antigen-binding sites. The fragments may include a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H) V_(L)). By using a linkerthat is too short to allow pairing between the two domains on the samechain, the domains are forced to pair with the complementary domains ofanother chain and create two antigen-binding sites. Techniques forgenerating diabodies are well known to those of skill in the art and arealso described in EP 404,097 and WO 93/11161, each specificallyincorporated herein by reference. Also, linear antibodies, which can bebispecific or monospecific, may include a pair of tandem Fd segments(V_(H) C_(H1)-V_(H) C_(H1)) that form a pair of antigen binding regionsmay be useful to the invention as described in Zapata et al. (1995), andincorporated herein by reference.

C. Compositions

The renal therapeutic agents contemplated herein can be expressed fromintact or truncated genomic or cDNA or from synthetic DNAs inprokaryotic or eukaryotic host cells by techniques well known to thoseof skill in the art. Exemplary host cells include, without limitation,prokaryotes including E. coli, or eukaryotes including yeast,Saccharomyces, insect cells, or mammalian cells, such as CHO, COS or BSCcells. One of ordinary kill in the art will appreciate that other hostcells can be used to advantage.

The term “construct” as used herein refers to a nucleic acid sequencecontaining at least one polynucleotide encoding a polypeptide of theinvention operably linked or fused to additional nucleic acids. Suchconstructs include vectors, plasmids, and expression cassettes encodingat least one polynucleotide encoding a polypeptide of the invention. Itis also envisioned that constructs could be polynucleotides encoding apolypeptide of the invention fused to other protein coding sequence togenerate fusion proteins as known to those of skill in the art.

Constructs can be inserted into mammalian host cells by methods known tothose of skill in the art including, but not limited to,electroporation, transfection, microinjection, micro-vessel transfer,particle bombardment, biolistic particle delivery, liposome mediatedtransfer and other methods described in Current Protocols in CellBiology, Unit 20, pub. John Wiley & Sons, Inc., 2004 and incorporatedherein by reference.

III. Therapeutic Uses

A. Subjects for Treatment

Renal therapeutic agents of the invention may be used in subjects thathave received renal injury, or those at risk of chronic renal failure.As used herein, a subject is said to be in, or at risk of, chronic renalfailure, or at risk of the need for renal replacement therapy (i.e.,chronic hemodialysis, continuous peritoneal dialysis, or kidneytransplantation), if the subject is reasonably expected to suffer aprogressive loss of renal function associated with progressive loss offunctioning nephron units. Whether a particular subject is in, or atrisk of, chronic renal failure is a determination which may routinely bemade by one of ordinary skill in the relevant medical or veterinary art.Subjects in, or at risk of, chronic renal failure, or at risk of theneed for renal replacement therapy, include but are not limited to thefollowing: subjects which may be regarded as afflicted with chronicrenal failure, end-stage renal disease, chronic diabetic nephropathy,hypertensive nephrosclerosis, chronic glomerulonephritis, hereditarynephritis, and/or renal dysplasia; subjects having a biopsy indicatingglomerular hypertrophy, tubular hypertrophy, chronic glomerulosclerosis,renal cell carcinoma, and/or chronic tubulointerstitial sclerosis;subjects having an ultrasound, MRI, CAT scan, or other non-invasiveexamination indicating renal fibrosis; subjects having an unusual numberof broad casts present in urinary sediment; subjects having a GFR whichis chronically less than about 50%, and more particularly less thanabout 40%, 30% or 20%, of the expected GFR for the subject; human malesubjects weighing at least about 50 kg and having a GFR which ischronically less than about 50 ml/min, and more particularly less thanabout 40 ml/min, 30 ml/min or 20 ml/min; human female subjects weighingat least about 40 kg and having a GFR which is chronically less thanabout 40 ml/min, and more particularly less than about 30 ml/min, 20ml/min or 10 ml/min; subjects possessing a number of functional nephronunits which is less than about 50%, and more particularly less thanabout 40%, 30% or 20%, of the number of functional nephron unitspossessed by a healthy but otherwise similar subject; subjects whichhave a single kidney; and subjects which are kidney transplantrecipients.

The methods and compositions of the present invention may be utilizedfor any mammalian subject. Such mammalian subjects include, but are notlimited to, human subjects or patients. Exemplary subjects may alsoinclude domesticated mammals (e.g., dogs, cats, horses), mammals withsignificant commercial value (e.g., dairy cows, beef cattle, sportinganimals), mammals with significant scientific value (e.g., captive orfree specimens of endangered species), or mammals which otherwise havevalue.

B. Excipients

The renal therapeutic agents of the invention, alone or conjugated, maybe formulated according to methods known to those skilled in the art toprepare pharmaceutically useful compositions, whereby the therapeuticagents are combined in a mixture with a pharmaceutically acceptablecarrier or excipient. A composition is said to be a “pharmaceuticallyacceptable carrier” if its administration can be tolerated by arecipient patient and preserves the activity of the active component, inthis case the renal therapeutic agent. Exemplary carriers include, butnot are limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The formulation should suit the modeof administration. Other suitable carriers are well known to thoseskilled in the art (see, REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed.,1995). Upon formulation, the antibody or immunoconjugate solutions willbe administered in a manner compatible with the dosage formulation andin such amount as is therapeutically effective.

C. Dosage

In general, the dosage of administered renal therapeutic agents willvary depending upon such factors as the patient's age, weight, height,sex, general medical condition and previous medical history. Forexample, it is typically desirable to provide the recipient with adosage of an antibody component, which is in the range of from about 1pg/kg to 10 mg/kg (amount of agent/body weight of patient), although alower or higher dosage also may be administered as circumstancesdictate. Range finding studies may be conducted to determine appropriatedosage by techniques known to those skilled in the art and as describedin Current Protocols in Pharmacology, Unit 10, pub. John Wiley & Sons,2003 and incorporated herein by reference. A skilled artisan willrecognize the therapeutically effective amount for each active compoundmay vary with factors including, but not limited to, the activity of thecompound used, stability of the active compound in the recipient's body,the total weight of the recipient treated, the route of administration,the ease of absorption, distribution, and excretion of the activecompound by the recipient, the age and sensitivity of the recipient tobe treated, the type of tissue, and the like.

For purposes of therapy, renal therapeutic agents are administered to apatient in a therapeutically effective amount in a pharmaceuticallyacceptable carrier. In this regard, a “therapeutically effective amount”is one that is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient patient. In the present context, an agent isphysiologically significant if its presence results in a clinicallysignificant improvement in an assay of renal function when administeredto a mammalian subject (e.g., a human patient). Such assays of renalfunction are well known to those of skill in the art and include,without being limited to, rates of increase in Blood Urea Nitrogen (BUN)levels, rates of increase in serum creatinine, static measurements ofBUN, static measurements of serum creatinine, glomerular filtrationrates (GFR), ratios of BUN/creatinine, serum concentrations of sodium(Na+), urine/plasma ratios for creatinine, urine/plasma ratios for urea,urine osmolality, daily urine output, and the like (see, Brenner andLazarus (1994), in HARRISON'S PRINCIPLES OF INTERNAL MEDICINE, 13thedition, Isselbacher et al., eds., McGraw Hill Text, N.Y.)

Additional pharmaceutical methods may be employed to control theduration of action of an antibody in a therapeutic application. Controlrelease preparations can be prepared through the use of polymers tocomplex or adsorb the renal therapeutic agent. For example,biocompatible polymers include matrices of poly(ethylene-co-vinylacetate) and matrices of a polyanhydride copolymer of a stearic aciddimer and sebacic acid (Sherwood et al., Bio/Technology 10:1446, 1992).The rate of release of an agent from such a matrix depends upon themolecular weight of the protein, the amount of agent within the matrix,and the size of dispersed particles (Saltzman et al., Biophys. J.55:163, 1989; Sherwood et al., Bio/Technology 10:1446, 1992). Othersolid dosage forms are described in REMINGTON'S PHARMACEUTICAL SCIENCES,19th ed, (1995) and can be prepared by techniques known to those skilledin the art.

D. Routes of Administration

Administration of renal therapeutic agents to a patient can beintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. When administeringtherapeutic proteins by injection, the administration may be bycontinuous infusion or by single or multiple boluses. Intravenousinjection provides a useful mode of administration due to thethoroughness of the circulation in rapidly distributing antibodies.

E. Methods for Testing Renal Therapeutic Agents

The renal therapeutic agents of the present invention may be tested inanimal models of chronic renal failure or nephronic degeneration.Mammalian models of nephronic degeneration in, for example, mice, rats,guinea pigs, cats, dogs, sheep, goats, pigs, cows, horses, and non-humanprimates, may be created by causing an appropriate direct or indirectinjury or insult to the renal tissues of the animal. For example, animalmodels of nephronic degeneration may be created by administeringcisplatin, which causes nephrotoxicity and reduced creatinine clearance.Animal models of nephronic degeneration may also be created byperforming a partial (e.g., ⅚) nephrectomy which reduces the number offunction nephron units to a level which initiates compensatory renalhypertrophy, further nephron loss, and the progressive decline in renalfunction (see, Vukicevic, et al. J. Bone Mineral Res. 2:533, 1987).Alternatively, animal models of renal cell carcinoma may be generated bysubcapsular renal injection of renal carcinoma (RENCA) cells thatresults in the development of primary tumors with subsequent developmentof metastases in the lungs, lymph nodes, and spleen (see, Hillman, G.G., Droz, J., and Haas, G. H. In Vivo, 8: 77-80, 1994). Theabove-described animal models may be generated by techniques well-knownto those of skill in the art.

The renal therapeutic agents may be administered to the above-describedanimal models and markers of renal function can be monitored (see,Examples 1-3). Preferably kidney function is determined using markers ofrenal function such as Blood Urea Nitrogen (BUN) levels, serumcreatinine levels, or glomerular filtration. Exemplary renal therapeuticagents will result in a decrease of BUN or serum creatinine levels orincrease in glomerular filtration rate compared to control animals.Control animals will be animal models treated with a control solutionnot containing the renal therapeutic agent being tested, preferably anon-irritating buffer solution or other carrier.

IV. Kits

The present invention provides articles of manufacture and kitscontaining materials useful for treating the pathological conditionsdescribed herein. The article of manufacture may include a container ofa medicament as described herein with a label. Suitable containersinclude, for example, bottles, vials, and test tubes. The containers maybe formed from a variety of materials such as glass or plastic. Thecontainer holds a composition having an active agent which is effectivefor treating, for example, diseases characterized by nephronicdegeneration. Alternatively, the container may hold a composition thatincludes a nephronic degeneration-inducing agent. The active agent inthe composition is a renal therapeutic agent of the invention, includinga peptide, protein, antibody, small molecule, or an agent such as avector or cell preparation capable of allowing production of a renaltherapeutic agent in vivo. The label on the container indicates that thecomposition is used for treating nephronic degenerative diseases, ormalignant diseases, and may also indicate directions for administrationand monitoring techniques, such as those described above.

The kit of the invention includes the container described above and asecond container, which may include a pharmaceutically acceptablediluent. It may further include other materials desirable from acommercial and user standpoint, including other buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

EXAMPLES

As can be appreciated from the disclosure provided above, the presentinvention has a wide variety of applications. Accordingly, the followingexamples are offered for illustration purposes and are not intended tobe construed as a limitation on the invention in any way. Those of skillin the art will readily recognize a variety of non-critical parametersthat could be changed or modified to yield essentially similar results.

Example 1 WISE/SOST Antibody Production

SOST and Wise both share the same gene structure, and produce a secretedprotein whose second exon encodes a cystein knot. Molecular dissectionof SOST at the amino acid level revealed putative LRP5/6 binding siteslocated in the first arm of the cystein knot. An immunoprecipitationassay of Flag tagged SOST variants and LRP6 was used to confirm which ofthese sites were necessary for LRP5/6 binding. Variants of SOST weregenerated with mutations at positions 60-62 (M1), 78-81 (M2), 89-90(M3), 100-103 (M4), 140-143 (M7), and 162-166 (Mss). Animmunoprecipitated western blot of Flag tagged SOST was mixed withLRP6-IgG and was quantified using phosphor-imager and its softwareImageQuant. SOST variants M1, M2 and M3 showed a significant loss ofbinding ability to LRP6 (FIG. 1), thus indicating potential sites formediating the block between SOST and its natural binding partnersincluding LRP5/6, BMP6, and BMP7.

In order to block the binding of SOST to LRP 5/6, BMP6, or BMP7inhibitory antibodies were generated that recognize the altered aminoacids of SOST variants M1, M2, and M3. Specific epitopes targeting theseamino acids were identified using antigenic hydrophobic plots. Theseplots revealed that the best sites for generation of an antibody werebetween amino acids 50-62, 68-80, and 83-98 of SEQ ID NO. 215 and 217.The resultant peptides selected as antigens to produce antibodies arelisted in SEQ ID NOS 15-18 and were used to generate monoclonalantibodies.

The peptides of SEQ ID NO 15-18 were used to immunize mice. Followingimmunization, B lymphocytes (B cells) were obtained from peripheralblood samples. The B cells from the immunized mice were then fused withmurine myeloma cells to produce hybridomas. The cells were cultured inHAT medium with hypoxanthine and thymidine to select out the hybridomacells. Hybridomas were then cultured by single-clone dilution inmicrotiter plates, followed by ELISA testing of the individual clonalsupernatants for desired anti-antigen reactivity. There were 27 ELISApositive monoclonal antibodies generated against SOST.

Example 2 Acute Renal Failure Treatment

Acute renal failure manifests itself immediately following renal insultor injury. Therapeutics of the present invention may be analyzed for useas a treatment for preventing or reversing acute renal failure.

Mice subjected to partial nephrectomies or decapsulation may be used asmodels of nephronic degeneration to test renal therapeutic agents of theinvention (see, Vukicevic, et al. J. Bone Mineral Res. 2:533, 1987). Apartial nephrectomy involves removing one kidney and ⅔ of the remainingkidney. After initial dramatic increases in plasma creatinine and BUNlevels indicating an acute failure phase, the levels decline to anelevated level compared to normal levels. Approximately two weeksfollowing surgery, the elevated level gradually increases with time asthe animal progresses to chronic renal failure. Decapsulation is a mocksurgery in which the kidneys are decapsulated but no renal tissue isremoved or nephronic injury introduced. Decapsulated mice may be used ascontrols for kidney functionality comparison.

To determine if a renal therapeutic agent of the invention can preventor delay the effects of acute renal failure, nephrectomized anddecapsulated mice that have immediately recovered from their respectivesurgeries may be used. Mice may be divided into six groups asfollows: 1) nephrectomized, receiving renal therapeutic agent; 2)nephrectomized, receiving vehicle buffer only; 3) nephrectomized,receiving no treatment; 4) decapsulated, receiving renal therapeuticagent; 5) decapsulated, receiving vehicle buffer only; and 6)decapsulated, receiving no treatment. Group one can be further dividedinto mice receiving 1, 3, 10, or 50 μg/kg body weight of renaltherapeutic agent. Prior to or during the acute failure phase,nephrectomized mice may be administered their respective treatment byintraperitoneal injection twice daily for at least three days. Serumcreatinine levels should be monitored prior to surgery, immediatelyfollowing surgery, each day of treatment, and for each of at least fourdays following the last injection.

A decrease in serum creatinine levels in nephrectomized mice treatedwith a therapeutic agent of the invention may indicate a successfulcandidate for further testing of preventing nephronic degeneration orinducing nephronic regeneration. An increase in serum creatinine levelsbeyond increases of serum creatinine levels of vehicle-only treated micemay indicate a therapeutic agent capable of inducing nephronicdegeneration. Such an agent may be useful in treating renal cellcarcinoma or other kidney cancer type.

Example 3 Chronic Renal Failure Treatment

Chronic renal failure manifests itself progressively following aninitial acute renal failure phase or renal insult without concomitantacute renal failure. Therapeutics of the present invention may beanalyzed for use as a treatment for preventing or reversing chronicrenal failure.

To determine if a therapeutic agent of the invention may prevent thedevelopment of chronic renal failure, nephrectomized and decapsulatedmice that have recovered from their respective surgeries for at leasttwo weeks may be used. Animals surviving the surgery for two weeks arepast the acute renal failure phase and have not yet entered chronicrenal failure.

Mice may be divided into six groups as follows: 1) nephrectomized,receiving renal therapeutic agent; 2) nephrectomized, receiving vehiclebuffer only; 3) nephrectomized, receiving no treatment; 4) decapsulated,receiving renal therapeutic agent; 5) decapsulated, receiving vehiclebuffer only; and 6) decapsulated, receiving no treatment. Group one canbe further divided into mice receiving 1, 3, 10, or 50 μg/kg body weightof renal therapeutic agent. Mice may be treated intraperitoneally atleast three times per week for a period of approximately 6-9 weeks.Serum creatinine levels should be monitored prior to treatment, duringthe treatment period, and at least 1 week following the treatmentperiod.

During weeks 1-5 of treatment, nephrectomized mice may exhibit elevatedserum creatinine levels compared to decapsulated mice. The amount ofelevation between the groups of nephrectomized mice may correlate withthe course of treatment used. If the serum creatinine levels are lesselevated with increasing amounts of the renal therapeutic agent beingtested, then the agent may be a successful candidate for further testsof preventing nephronic degeneration and inducing nephronicregeneration. If the serum creatinine levels become increasinglyelevated with increasing amounts of the renal therapeutic agent indecapsulated mice, then the agent may be a nephronic degenerationinducing agent. Such an agent may be useful in treating renal cellcarcinoma or other kidney cancer type.

Example 4 Renal Cell Carcinoma Treatment

Constitutive activation of the Wnt signaling pathway may be involved inthe development of renal cell carcinoma and other kidney cancer types.The renal therapeutic agents of the invention that result in ectopicactivation of the Wnt signaling pathway via interaction with the naturalreceptors of WISE and SOST may be useful in therapies treating renalcell carcinoma or other kidney cancer types. Therapeutics of the presentinvention may be analyzed for use as a treatment for preventing orreversing kidney cancer types.

To investigate novel therapeutic strategies for the treatment of humanrenal cell carcinoma, such as adoptive immunotherapy or cytokinetherapy, murine renal cell carcinoma has been a particularly suitableanimal model for assessing novel therapeutic approaches (Sayers, T. J.,Wiltrout, T. A., McCormick, K., Husted, C., and Wiltrout, R. H., CancerRes., 50: 5414-5420, 1990; Salup, R. R., and Wiltrout, R. H. CancerRes., 46: 3358-3363, 1986). In this model, primary kidney tumors areinduced by subcapsular renal injection of renal carcinoma (RENCA) cellswith subsequent development of metastases in the lungs, lymph nodes, andspleen (Hillman, G. G., Droz, J., and Haas, G. H. In Vivo, 8: 77-80,1994).

Murine RENCA cells originally obtained from a tumor that arosespontaneously in the kidney of BALB/c mice may be injected into BALB/cmice to generate a renal cell carcinoma model. Histologically, RENCA isa granular cell type adenocarcinoma, which is pleomorphic with largenuclei. Monolayers of murine RENCA cells may be grown in RPMI 1640 withphenol red supplemented with 10% FCS, 2 mM L-glutamine, 100 unitspenicillin/ml, and 100 μg of streptomycin/ml. RENCA cells may becultured in a humidified atmosphere of 95% air and 5% carbon dioxide at37° C.

Female BALB/c mice approximately 6-8 weeks of age (approximate weight,20 g) may be injected with RENCA cells in 0.2-ml aliquots into thesubcapsular space of the left kidney performed through a flank incisionafter the animals are anesthetized with 0.5-1.5 volume percentisoflurane, which may be used in combination with an oxygen flow of 1.5l/min. The subcapsular renal injection of RENCA cells in a syngeneicBALB/c mouse may be followed by the progressive development of a primarytumor mass in the left kidney. One week after application, the primarytumor may be macroscopically visible; after 10 days, spontaneousmetastases may develop in the regional lymph nodes, in the lung, theperitoneum, and the liver, allowing the RENCA model to be stagedsimilarly to human renal cell carcinoma. The mean survival time ofRENCA-bearing mice may be 32 days after RENCA cells are injected.

Treatments with a renal therapeutic agent of the invention or vehicleonly may be initiated 1 day after tumor cell inoculation into thesubcapsular space of the left kidney. Mice receiving the renaltherapeutic agent may receive about 1, 3, 10, or 50 μg/kg body weight ofthe renal therapeutic agent intraperitoneally at least three times perweek for a period of approximately 6-9 weeks. Serum creatinine levelsshould be monitored prior to treatment, during the treatment period, andat least 1 week following the treatment period. Animal weights should betaken every other day.

Two or 3 weeks after starting treatment, 6 or 10 mice, respectively, maybe sacrificed in each group for determination of weight and volume ofprimary tumors, weight, and number of metastasis of the lung andmetastasis formation in the abdominal lymph nodes. The volumes ofprimary tumors taken macroscopically may be calculated by taking andmultiplying the distances of all three dimensions. The number ofmetastases in the lung and abdominal lymph nodes may be counted using adissection microscope. In the abdominal cave, all visible lymph nodesmay be counted for detection of metastasis, knowing that in healthyanimals visible lymph nodes are usually absent. More animals may besacrificed at later time points to monitor the progression or regressionof tumor development.

A renal therapeutic agent of the invention that results in a significantdecrease in primary tumor size or number of metastasis compared to micetreated with vehicle only may be successful candidates for renal cellcarcinoma therapy. Agents that do not result in a significant decreaseor result in a significant increase in primary tumor size or number ofmetastasis may be successful candidates for preventing nephronicdegeneration or promoting nephronic regeneration.

1-20. (canceled)
 21. An isolated antibody that specifically binds to 5,8, 10, or 15 contiguous amino acids of SEQ ID NO:
 171. 22. The isolatedantibody of claim 21, wherein the antibody specifically binds to 5contiguous amino acids of SEQ ID NO:
 171. 23. The isolated antibody ofclaim 21, wherein the antibody specifically binds to 8 contiguous aminoacids of SEQ ID NO:
 171. 24. The isolated antibody of claim 21, whereinthe antibody specifically binds to 10 contiguous amino acids of SEQ IDNO:
 171. 25. The isolated antibody of claim 21, wherein the antibodyspecifically binds to 15 contiguous amino acids of SEQ ID NO:
 171. 26.The isolated antibody of claim 22, wherein the antibody is selected fromthe group consisting of a monoclonal antibody, a polyclonal antibody, aFv fragment of an antibody, a Fab fragment of an antibody, a F(ab′)₂fragment of an antibody, a recombinant single chain Fv fragment (scFv),a diabody, a triabody, and a tetrabody.
 27. The isolated antibody ofclaim 26, wherein the antibody is a monoclonal antibody.